Modified MIC Method for Cationic Antimicrobial Peptides

This method is based on the classical microtitre broth dilution recommended by the National Committee of Laboratory Safety and Standards (NCLSS) as published in Amsterdam, D. 1996. Susceptibility testing of Antimicrobials in liquid media. In “Antibiotics in Laboratory Medicine”, Lorian, V., ed. Fourth Edition, pp.52-111. Williams and Wilkins, Baltimore. The modifications were first introduced by Intrabiotics Inc at the 1996 ICAAC, and recommended for general use by Bob Hancock at the First Gordon Conference on Antimicrobial Peptides. They are included here in the hope that they will be used standardly by workers in the field for antimicrobial susceptibility testing to permit comparisons to be made between the data of different labs. We have found this method to be reliable and reproducible.

Below we have indicated some results from our comparisons of different methods.

Materials:

It is important that you use the materials mentioned below. For example, do not substitute polystyrene for polypropylene microtitre plates. Cationic peptides bind polystyrene (especially “tissue culture treated” polystyrene).

1. Sterile tubes (16 X 125 mm) containing 5 ml of Mueller Hinton Broth (MHB, Cat. No. 0757-17-6, DIFCO)
2. Mueller Hinton agar plates (MHA)
3. Sterile 96-well polypropylene microtitre plates (COSTAR Cat. No. 3790, distrubuted as FISHER Cat. No. CS003790 or SIGMA Cat. No. M4404)
4. Polypropylene microcentrifuge tubes (Cat. No. 000-MICR-150, Elkay Products, Inc.) or glass tubes coated with Sigmacote
5. Sterile petri dishes
6. Test peptides quantated by amino acid analysis
7. 0.01% acetic acid (Cat. No. A-38-4, FISHER) containing 0.2% bovine serum albumin (BSA, Cat. No. 735086, Boehringer Mannheim GmbH)
8. 0.02% acetic acid containing 0.4% BSA

Methods:

1. Inoculate 5 ml MHB in tubes with test strains from MHA plates and grow overnight at 37^oC on a shaker (180 rpm).
2. Make serial dilutions of test peptides (at 10 times the required test concentrations) in 0.01% acetic acid, 0.2% BSA in polypropylene or coated glass tubes:
   1. dissolve test peptide in dH₂O at 20 times the required maximal concentration (enough final volume for all tests to be performed on a given day);
   2. dilute it into an equal volume of 0.02% acetic acid, 0.4% BSA to get 10 times the required maximal concentration;
   3. then do serial doubling dilutions in 0.01% acetic acid, 0.2% BSA to get serial dilutions of peptides at 10 times the required test concentrations, eg., 640, 320, 160, …2.5 µg/ml.
3. Dilute overnight bacterial cultures in MHB to give 2 – 7 x 10⁵ colony forming units/ml.
4. Dispense 100 µl of bacterial suspension in each well from column 1 to column 11. Do not add bacteria to column 12, and instead dispense 100 µl of MHB (sterility control and blank for the plate scanner).
5. Add 11 µl of 10x test peptide each well from column 1 to column 10 (column 11 is a control for bacteria alone, with no peptide).
6. Incubate the plates at 37^oC for 18-24 hours. Check again at 40-48 hours.
7. MIC can be taken as the lowest concentration of drug that reduces growth by more than 50%.
8. Plate l0 µl 10^-6 dilution of overnight cultures on MHA plates to determine a viable count. The MBC (Minimal bactericidal concentration) can be determined by plating out the contents of the first 3 wells showing no visible growth of bacteria onto MHA plates and incubating at 37^oC for 18 hr. The lowest concentration of peptide that prevents any residual colony formation is the MBC.

Effect of MIC determination method on the MIC value recorded. Methods 1 and 2 are the methods given above and method 6 is the broth microdilution method of Amsterdam (see above reference).

* “tissue culture treated”